Abstract: Genomic DNA was extracted by advanced CTAB methods from Medicago sativa and Medicago hispida. The optimum SCoT-PCR (start codon targeted polymorphism) reaction system in alfalfa was established with orthogonal design experiments on four levels of five factors (DNA, dNTPs, Mg²⁺, Taq polymerase, and primer). A suitable SCoT reaction system was a 20 μL mixture containing 50 ng DNA, 1.00 mmol/L Mg²⁺, 1.20 U Taq polymerase, 0.40 mmol/L dNTPs and 0.30 μmol/L primers. Of the 50 primers screened, 13 generated highly polymorphic fragments useable as SCoT markers, and 34 accessions were amplified by these 13 primers. A total of 103 bands (including 92 polymorphic bands) were detected by the 13 chosen primers. The 34 accessions were divided into two groups. Two M. hispida cultivars from Anhui and Jiangsu were clustered into one group. The remaining 32 cultivars were classified into three sub-groups with a similarity coefficient of 0.757. Fall dormancy alfalfa cultivars were mostly distributed in the second sub-group. Semi-fall dormancy and non-fall dormancy alfalfa cultivars did not converge into the same sub-group thus showing incomplete similarity between fall dormancy alfalfas.